重组Anti-LOX抗体[EPR4025] (ab174316)

Flow Cytometry (Intracellular) analysis of ab174316. HeLa (Human cervix adenocarcinoma epithelial cell) cells were fixed in 4% paraformaldehyde and permeabilized with 90% methanol. ab174316 was used at 1:100 dilution (1ug) (Red). Secondary antibody Goat anti rabbit IgG (Alexa Fluor® 647, ab150083) at 1:5000 dilution. Isotype control Rabbit monoclonal IgG (ab172730) (Black). Unlabelled control, cells without incubation with primary antibody and secondary antibody (Blue).

False colour image of Western blot: Anti-LOX antibody [EPR4025] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab174316 was shown to bind specifically to LOX. A band was observed at 52 kDa in wild-type HeLa cell lysates with no signal observed at this size in Lox knockout cell line ab261801 (knockout cell lysate ab256981). To generate this image, wild-type and Lox knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Primary antibody dilution:

ab174316 Anti-LOX antibody 1:5000 dilution (0.1mg/ml)

ab179483 Anti-HIF-1 alpha antibody 1:500 dilution (0.3mg/ml)

Blocking buffer / Dilution buffer and concentration:

5% NFDM/TBST

LOX and HIF-1 alpha could be induced under hypoxia condition (PMID: 23545606, PMID: 30226558).

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded human kidney with purified ab174316 at a working dilution of 1 in 900. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

Immunofluorescence staining of Jurkat cells with purified ab174316 at a working dilution of 1 in 300, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti rabbit, used at a dilution of 1 in 200. The cells were fixed in 4% PFA.

Immunocytochemistry/ Immunofluorescence analysis of formaldehyde-fixed Triton X-100 permeabilized human chondrocytes staining with ab174316 at 1/100 dilution in PBS with 0.1% triton x100. Secondary antibody was Alexa Fluor^® 488 Goat Anti-Rabbit at 1/2000 dilution. Samples were incubated with the primary antibody for 30 minutes at 37°C. Blocking was with 0.1% BSA for 30 minutes at 37°C (ref: PMC7204390)

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Western blot analysis on immunoprecipitation pellet from (Lane 1) WI-38 (Human fetal lung fibroblast cell line) cells lysate or (Lane 2) 1X PBS (negative control) using unpurified ab174316 at a 1/10 dilution and HRP-conjugated anti-rabbit IgG preferentially detecting the non-reduced form of rabbit IgG.